The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions that afflicts almost 200,000 people a year in the US with 75,000 deaths. The hallmark of ALI is inflammation-induced disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature, resulting in respiratory failure from the leakage of fluid, protein, and cells into th airspaces of the lung. EC barrier function is regulated by a balance between barrier-disrupting cellular contractile forces and barrier-protective tethering forces, with both competing forces linked to the EC actin cytoskeleton by a variety binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in regulating the cytoskeletal structures hat determine EC permeability. The central hypothesis of this proposal is that pulmonary vascular leak in ALI is regulated by cortactin function at the level of lung EC. With this background, studies are proposed using integrated approaches across scientific disciplines to examine the contribution of cortactin to ALI pathogenesis in vitro and in vivo. Specific Aim #1 will characterize the functional effects of cortactin on pulmonary EC cytoskeletal structure and permeability in vitro. The effects of cortactin deficiency (via siRNA) and overexpression on the following parameters will be determined in cultured human pulmonary EC: 1) lamellipodia structure and dynamics, 2) cortical actin structure, 3) junctional complex structure, 4) permeability. Multiple modalities will be employed, including confocal microscopy, live cell imaging, kymography, novel atomic force microscopy measurements of junctional complex adhesive strength and cell elastic properties, biochemical assays, and in vitro models of EC permeability. Specific Aim #2 will determine the association of cortactin single nucleotide polymorphisms (SNPs) with inflammatory lung injury and their functional effects on pulmonary EC. Preliminary studies by our group have identified promoter and coding SNPs in the cortactin gene that are associated with ALI in patients. Validation of ALI-association in a replicate cohort will be performed for SNPs in the CTTN promoter and coding region, and their effects on cortactin expression and lung endothelial function in vitro will be characterized as outlined in SA#1. Specific Aim #3 will transnationally characterize the effects of cortactin and disease-associated cortactin SNPs on inflammatory lung injury in mice. Two models of ALI in mice will be employed: 1) intratracheal LPS; 2) ventilator-induced lung injury. The effects of decreased cortactin expression in these models will be determined using cortactin +/- mice and in vivo siRNA, while the effects of overexpression of wild type and SNP cortactin in mouse lungs will be explored using ACE- antibody targeted liposomal delivery. An EC-specific conditional cortactin knockout mouse will be generated. These studies will mechanistically characterize the important role of cortactin in the pathogenesis of ALI and provide new insights into potential therapeutic approaches to prevent or reduce vascular leak.